ONCOLOGY REPORTS 32: 159-166, 2014
YKL-40 downregulation is a key factor to overcome temozolomide resistance in a glioblastoma cell line YASUTO AKIYAMA1,3*, TADASHI ASHIZAWA1*, MASARU KOMIYAMA1, HARUO MIYATA1, CHIE OSHITA1, MAHO OMIYA1, AKIRA IIZUKA1, AKIKO KUME1, TAKASHI SUGINO2, NAKAMASA HAYASHI3, KOICHI MITSUYA3, YOKO NAKASU3 and KEN YAMAGUCHI1 1
Immunotherapy Division, Shizuoka Cancer Center Research Institute; Divisions of 2Pathology and 3Neurosurgery, Shizuoka Cancer Center Hospital, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8777, Japan Received March 6, 2014; Accepted April 22, 2014 DOI: 10.3892/or.2014.3195
Abstract. The frequent recurrence of glioblastoma multiforme (GBM) after standard treatment with temozolomide (TMZ) is a crucial issue to be solved in the clinical field. O 6 ‑methylguanine‑DNA methyltransferase (MGMT) is considered one of the major mechanisms involved in TMZ resistance. However, some important mechanisms for TMZ resistance other than MGMT have recently been identified. In the present study, we established a TMZ-resistant (TMZ-R) U87 glioblastoma cell line in vitro and in vivo and investigated novel targeting molecules other than MGMT in those cells. The TMZ-R U87 glioblastoma cell line was established in vitro and in vivo. TMZ-R U87 cells showed a more invasive activity and a shorter survival time in vivo. Gene expression analysis using DNA microarray and quantitative PCR (qPCR) demonstrated that YKL‑40, MAGEC1 and MGMT mRNA expression was upregulated 100-, 83- and 6-fold, respectively in the TMZ-R U87 cell line. Western blot analysis and qPCR demonstrated that STAT3 phosphorylation, STAT3 target genes and stem cell and mesenchymal marker genes were upregulated to a greater extent in the TMZ‑resistant cell line. Notably, short hairpin (sh)RNA‑based inhibition against the YKL‑40 gene resulted in moderate growth inhibition in the
Correspondence to: Dr Yasuto Akiyama, Immunotherapy Division, Shizuoka Cancer Center Research Institute, 1007 Shimonagakubo, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8777, Japan E-mail: [email protected] *
Contributed equally
Abbreviations: GBM, glioblastoma multiforme; TMZ, temozolomide; STAT, signal transducer and activator of transcription; SH, Src homology; DMSO, dimethyl sulfoxide; JAK, Janus kinase; T/C, tumor/control; siRNA, small interfering RNA; shRNA, small hairpin RNA
Key words: TMZ resistance, STAT3, YKL-40, shRNA, recurrent glioblastoma
resistant cells in vitro and in vivo. Additionally, YKL‑40 gene inhibition exhibited significant suppression of the invasive activity and particularly partially restored the sensitivity to TMZ. Therefore, YKL‑40 may be a novel key molecule in addition to MGMT, that is responsible for TMZ resistance in glioblastoma cell lines and could be a new target to overcome TMZ resistance in recurrent glioblastomas in the future. Introduction Glioblastoma multiforme (GBM) is the most malignant and aggressive tumor, and GBM patients have an extremely poor prognosis and a mean survival time of less than 2 years, even following treatment with recent concomitant chemoradiation (1,2). Temozolomide (TMZ) is an alkylating agent that induces DNA methylation of guanine at the O6 position and triggers mismatch repair, which leads to arrest of the cell cycle and apoptosis (3). O6‑methylguanine‑DNA methyltransferase (MGMT) is well known for removing methyl groups from the O6 position of guanine and contributing to TMZ resistance induction (4). Several clinical studies have demonstrated that high MGMT expression through the methylation of the MGMT promoter is one of the genuine mechanisms responsible for TMZ resistance. Thus, novel therapeutics that aim to suppress TMZ resistance by deleting MGMT have been pursued, and O6‑benzylguanine has been considered to be a promising therapeutic candidate. However, clinical trials did not show the restoration of TMZ resistance (5,6). Considering that no tools presently exist to treat TMZ‑resistant (TMZ‑R) recurrent glioblastoma, novel therapeutic approaches that regulate MGMT expression and restore the sensitivity to TMZ are highly required. Moreover, it has been suggested based on multi-omic analysis that mechanisms other than MGMT may trigger TMZ resistance. Several novel biomarkers that are linked to MGMT expression and methylation status, such as the HOX signature and EGFR expression (7), somatic mutations of the mismatch repair gene MSH6 (8), prolyl 4‑hydroxylase, β polypeptide (P4HB) (9), mutated EGFR (EGFRvIII) (10) and CD74 (11), have been reported. Regarding novel approaches to overcome MGMT‑related resistance to TMZ, bortezomib as a proteasome inhibitor (12), telomerase inhibition (13), a combination of
160
AKIYAMA et al: YKL-40 DOWNREGULATION IS A KEY FACTOR FOR OVERCOMING TMZ RESISTANCE
interleukin (IL)‑24 with TMZ (14) and inactivation of MGMT by gene therapy (15) have been demonstrated to show moderate effects on MGMT downregulation and tumor cell death. According to a recent molecular classification study of various glioblastomas, it is known that a mesenchymal signature expressing STAT3 and the C/EBPβ genes is closely linked to poor prognosis and TMZ resistance with tumor recurrence (16-19). These genes could be new possible targets for overcoming TMZ resistance in GB. In contrast, MGMT is not classified into any specific subtype marker, including the mesenchymal signature (17). In the present study, we identified the YKL‑40 and MAGEC1 genes as TMZ resistance‑associated biomarkers in the TMZ‑R U87 cell line, which showed an obvious resistance in vivo. Furthermore, we focused on the YKL‑40 and STAT3 mechanisms and proposed a restoration model of TMZ resistance. Materials and methods Cell lines. The human glioblastoma cell lines LN18, T98G and U87 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen), penicillin and streptomycin. Antibodies and reagents. Antibodies against STAT3, phospho‑specific STAT3 (Tyr705), MGMT and β-actin were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) and Becton‑Dickinson (BD) Biosciences (Franklin Lakes, NJ, USA) for western blotting (WB). A mouse antihuman YKL‑40 monoclonal antibody (MoAb) was purchased from Abcam (Cambridge, MA, USA) for immunohistochemical (IHC) studies. Short hairpin (sh)RNAs specific for the human STAT3 or YKL‑40 genes were purchased from Qiagen GmbH (Hilden, Germany). TMZ was purchased from Sigma‑Aldrich (St. Louis, MO, USA) and was suspended in sterile 0.5% w/v methyl cellulose 400cp solution (Wako, Tokyo, Japan). Establishment of the TMZ‑resistant U87 cell line. The U87 parental cell line, which is sensitive to TMZ, was first maintained in low doses of TMZ (5 µM) and then successively exposed to incremental doses of TMZ (up to 150 µM). After the killing of a majority of the cells, the surviving cells were maintained until a normal rate of growth was obtained. The IC50 value of TMZ was evaluated using the WST‑1 assay. Cell proliferation assay. Cell proliferation was examined using the WST‑1 assay (Dojindo Laboratories, Kumamoto, Japan) as described previously (20). Briefly, 1-2x104 human glioma cells were seeded into each well of a 96‑well microculture plate (Corning, NY, USA). After 4 days, the WST‑1 substrate was added to the culture and the optical density (OD) was measured at 450 and 620 nm using an immunoreader (Immuno Mini NJ-2300, Nalge Nunc International, Roskilde, Denmark). The IC50 value was defined as the dose required for a 50% reduction in the OD calculated from the survival curve. Percent survival was calculated as follows: (mean OD of test wells - mean OD of background wells)/(mean OD of control wells - mean OD of background wells).
DNA microarray analysis. Total RNA from the U87 parental and TMZ‑R cell lines was extracted using the NucleoSpin RNA II kit (Takara Bio Inc., Shiga, Japan) according to the manufacturer's instructions. One microgram of total RNA, which was qualified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), was amplified to 100 µg of cRNA and hybridized to a high‑density oligonucleotide array (GeneChip Human Genome U133 Plus 2.0 array; Affymetrix, Santa Clara, CA, USA). The intensity for each feature of the array was calculated using the GeneSpringGX ver11 (Agilent) software. To calculate the change in the average intensity, normalization for all probe sets was performed. Genes whose expression was significantly altered by >5‑fold at the 5th and 20th passages of the TMZ-R U87 cell line compared to the U87 parental cell line were analyzed. Inhibition of STAT3 or YKL‑40 gene expression using shRNA transfection of the TMZ‑resistant U87 cells. shRNA gene transfection into the TMZ‑R U87 cell line was performed using a lipofection FreeStyle MAX reagent (Life Technologies, Carlsbad, CA, USA). Four micrograms of plasmid at 1 mg/ml containing STAT3 or YKL‑40 shRNA (SureSilencing shRNA vector; Qiagen) and the same dose of FreeStyle MAX reagent were suspended in 100 µl of Opti‑MEM I reduced‑serum medium (Life Technologies), which was then mixed and incubated for 15 min at room temperature (RT). The solution was added to 2x106 TMZ‑R U87 cells and incubated at 37˚C for 1 h. After washing, the cells were incubated in DMEM + 10% FBS, harvested on day 3 of culture and utilized for in vitro and in vivo experiments. Cell invasion assay. Invasion assays using parental U87 and TMZ‑R U87 cells were performed using Matrigel‑coated (0.33 mg/ml) Transwell inserts with a 8‑µm pore size (BD Biosciences, Franklin Lakes, NJ, USA). Cells at 1x105/ml (500 µl) were added to Transwells in triplicate, and 750 µl of DMEM containing 10% FBS was added to the lower wells. After 12-18 h of incubation, the cells that invaded through the membrane were fixed and stained with Diff-Quik II solution (Dade Behring AG, Germany). Migrated cells were counted using microscopy. Quantitative polymerase chain reaction (qPCR) analysis. Real-time PCR analysis of 10 genes that were rated as significantly changed at the expression level (>10‑fold, P
YKL-40 downregulation is a key factor to overcome temozolomide resistance in a glioblastoma cell line YASUTO AKIYAMA1,3*, TADASHI ASHIZAWA1*, MASARU KOMIYAMA1, HARUO MIYATA1, CHIE OSHITA1, MAHO OMIYA1, AKIRA IIZUKA1, AKIKO KUME1, TAKASHI SUGINO2, NAKAMASA HAYASHI3, KOICHI MITSUYA3, YOKO NAKASU3 and KEN YAMAGUCHI1 1
Immunotherapy Division, Shizuoka Cancer Center Research Institute; Divisions of 2Pathology and 3Neurosurgery, Shizuoka Cancer Center Hospital, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8777, Japan Received March 6, 2014; Accepted April 22, 2014 DOI: 10.3892/or.2014.3195
Abstract. The frequent recurrence of glioblastoma multiforme (GBM) after standard treatment with temozolomide (TMZ) is a crucial issue to be solved in the clinical field. O 6 ‑methylguanine‑DNA methyltransferase (MGMT) is considered one of the major mechanisms involved in TMZ resistance. However, some important mechanisms for TMZ resistance other than MGMT have recently been identified. In the present study, we established a TMZ-resistant (TMZ-R) U87 glioblastoma cell line in vitro and in vivo and investigated novel targeting molecules other than MGMT in those cells. The TMZ-R U87 glioblastoma cell line was established in vitro and in vivo. TMZ-R U87 cells showed a more invasive activity and a shorter survival time in vivo. Gene expression analysis using DNA microarray and quantitative PCR (qPCR) demonstrated that YKL‑40, MAGEC1 and MGMT mRNA expression was upregulated 100-, 83- and 6-fold, respectively in the TMZ-R U87 cell line. Western blot analysis and qPCR demonstrated that STAT3 phosphorylation, STAT3 target genes and stem cell and mesenchymal marker genes were upregulated to a greater extent in the TMZ‑resistant cell line. Notably, short hairpin (sh)RNA‑based inhibition against the YKL‑40 gene resulted in moderate growth inhibition in the
Correspondence to: Dr Yasuto Akiyama, Immunotherapy Division, Shizuoka Cancer Center Research Institute, 1007 Shimonagakubo, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8777, Japan E-mail: [email protected] *
Contributed equally
Abbreviations: GBM, glioblastoma multiforme; TMZ, temozolomide; STAT, signal transducer and activator of transcription; SH, Src homology; DMSO, dimethyl sulfoxide; JAK, Janus kinase; T/C, tumor/control; siRNA, small interfering RNA; shRNA, small hairpin RNA
Key words: TMZ resistance, STAT3, YKL-40, shRNA, recurrent glioblastoma
resistant cells in vitro and in vivo. Additionally, YKL‑40 gene inhibition exhibited significant suppression of the invasive activity and particularly partially restored the sensitivity to TMZ. Therefore, YKL‑40 may be a novel key molecule in addition to MGMT, that is responsible for TMZ resistance in glioblastoma cell lines and could be a new target to overcome TMZ resistance in recurrent glioblastomas in the future. Introduction Glioblastoma multiforme (GBM) is the most malignant and aggressive tumor, and GBM patients have an extremely poor prognosis and a mean survival time of less than 2 years, even following treatment with recent concomitant chemoradiation (1,2). Temozolomide (TMZ) is an alkylating agent that induces DNA methylation of guanine at the O6 position and triggers mismatch repair, which leads to arrest of the cell cycle and apoptosis (3). O6‑methylguanine‑DNA methyltransferase (MGMT) is well known for removing methyl groups from the O6 position of guanine and contributing to TMZ resistance induction (4). Several clinical studies have demonstrated that high MGMT expression through the methylation of the MGMT promoter is one of the genuine mechanisms responsible for TMZ resistance. Thus, novel therapeutics that aim to suppress TMZ resistance by deleting MGMT have been pursued, and O6‑benzylguanine has been considered to be a promising therapeutic candidate. However, clinical trials did not show the restoration of TMZ resistance (5,6). Considering that no tools presently exist to treat TMZ‑resistant (TMZ‑R) recurrent glioblastoma, novel therapeutic approaches that regulate MGMT expression and restore the sensitivity to TMZ are highly required. Moreover, it has been suggested based on multi-omic analysis that mechanisms other than MGMT may trigger TMZ resistance. Several novel biomarkers that are linked to MGMT expression and methylation status, such as the HOX signature and EGFR expression (7), somatic mutations of the mismatch repair gene MSH6 (8), prolyl 4‑hydroxylase, β polypeptide (P4HB) (9), mutated EGFR (EGFRvIII) (10) and CD74 (11), have been reported. Regarding novel approaches to overcome MGMT‑related resistance to TMZ, bortezomib as a proteasome inhibitor (12), telomerase inhibition (13), a combination of
160
AKIYAMA et al: YKL-40 DOWNREGULATION IS A KEY FACTOR FOR OVERCOMING TMZ RESISTANCE
interleukin (IL)‑24 with TMZ (14) and inactivation of MGMT by gene therapy (15) have been demonstrated to show moderate effects on MGMT downregulation and tumor cell death. According to a recent molecular classification study of various glioblastomas, it is known that a mesenchymal signature expressing STAT3 and the C/EBPβ genes is closely linked to poor prognosis and TMZ resistance with tumor recurrence (16-19). These genes could be new possible targets for overcoming TMZ resistance in GB. In contrast, MGMT is not classified into any specific subtype marker, including the mesenchymal signature (17). In the present study, we identified the YKL‑40 and MAGEC1 genes as TMZ resistance‑associated biomarkers in the TMZ‑R U87 cell line, which showed an obvious resistance in vivo. Furthermore, we focused on the YKL‑40 and STAT3 mechanisms and proposed a restoration model of TMZ resistance. Materials and methods Cell lines. The human glioblastoma cell lines LN18, T98G and U87 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen), penicillin and streptomycin. Antibodies and reagents. Antibodies against STAT3, phospho‑specific STAT3 (Tyr705), MGMT and β-actin were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA) and Becton‑Dickinson (BD) Biosciences (Franklin Lakes, NJ, USA) for western blotting (WB). A mouse antihuman YKL‑40 monoclonal antibody (MoAb) was purchased from Abcam (Cambridge, MA, USA) for immunohistochemical (IHC) studies. Short hairpin (sh)RNAs specific for the human STAT3 or YKL‑40 genes were purchased from Qiagen GmbH (Hilden, Germany). TMZ was purchased from Sigma‑Aldrich (St. Louis, MO, USA) and was suspended in sterile 0.5% w/v methyl cellulose 400cp solution (Wako, Tokyo, Japan). Establishment of the TMZ‑resistant U87 cell line. The U87 parental cell line, which is sensitive to TMZ, was first maintained in low doses of TMZ (5 µM) and then successively exposed to incremental doses of TMZ (up to 150 µM). After the killing of a majority of the cells, the surviving cells were maintained until a normal rate of growth was obtained. The IC50 value of TMZ was evaluated using the WST‑1 assay. Cell proliferation assay. Cell proliferation was examined using the WST‑1 assay (Dojindo Laboratories, Kumamoto, Japan) as described previously (20). Briefly, 1-2x104 human glioma cells were seeded into each well of a 96‑well microculture plate (Corning, NY, USA). After 4 days, the WST‑1 substrate was added to the culture and the optical density (OD) was measured at 450 and 620 nm using an immunoreader (Immuno Mini NJ-2300, Nalge Nunc International, Roskilde, Denmark). The IC50 value was defined as the dose required for a 50% reduction in the OD calculated from the survival curve. Percent survival was calculated as follows: (mean OD of test wells - mean OD of background wells)/(mean OD of control wells - mean OD of background wells).
DNA microarray analysis. Total RNA from the U87 parental and TMZ‑R cell lines was extracted using the NucleoSpin RNA II kit (Takara Bio Inc., Shiga, Japan) according to the manufacturer's instructions. One microgram of total RNA, which was qualified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), was amplified to 100 µg of cRNA and hybridized to a high‑density oligonucleotide array (GeneChip Human Genome U133 Plus 2.0 array; Affymetrix, Santa Clara, CA, USA). The intensity for each feature of the array was calculated using the GeneSpringGX ver11 (Agilent) software. To calculate the change in the average intensity, normalization for all probe sets was performed. Genes whose expression was significantly altered by >5‑fold at the 5th and 20th passages of the TMZ-R U87 cell line compared to the U87 parental cell line were analyzed. Inhibition of STAT3 or YKL‑40 gene expression using shRNA transfection of the TMZ‑resistant U87 cells. shRNA gene transfection into the TMZ‑R U87 cell line was performed using a lipofection FreeStyle MAX reagent (Life Technologies, Carlsbad, CA, USA). Four micrograms of plasmid at 1 mg/ml containing STAT3 or YKL‑40 shRNA (SureSilencing shRNA vector; Qiagen) and the same dose of FreeStyle MAX reagent were suspended in 100 µl of Opti‑MEM I reduced‑serum medium (Life Technologies), which was then mixed and incubated for 15 min at room temperature (RT). The solution was added to 2x106 TMZ‑R U87 cells and incubated at 37˚C for 1 h. After washing, the cells were incubated in DMEM + 10% FBS, harvested on day 3 of culture and utilized for in vitro and in vivo experiments. Cell invasion assay. Invasion assays using parental U87 and TMZ‑R U87 cells were performed using Matrigel‑coated (0.33 mg/ml) Transwell inserts with a 8‑µm pore size (BD Biosciences, Franklin Lakes, NJ, USA). Cells at 1x105/ml (500 µl) were added to Transwells in triplicate, and 750 µl of DMEM containing 10% FBS was added to the lower wells. After 12-18 h of incubation, the cells that invaded through the membrane were fixed and stained with Diff-Quik II solution (Dade Behring AG, Germany). Migrated cells were counted using microscopy. Quantitative polymerase chain reaction (qPCR) analysis. Real-time PCR analysis of 10 genes that were rated as significantly changed at the expression level (>10‑fold, P
